In 2008 we continued our accrual of patients into our blister study protocol. This experimental protocol involves the formation of blisters on the forearms of volunteers and the study of the inflammatory response in vivo in these blisters. In addition to several normals, we have also studied three patients with chronic graft-versus-host disease and two patients with Jobs Syndrome. Additional patients will be required to fully power these studies to determine whether the observed dysregulation of cytokine production is statistically significant (Kol Zarember, 20% effort). [unreadable] [unreadable] In 2008 we completed our studies of fibrinogen as a regulator of IL8 production. In previous work we showed that fibrinogen amplifies IL8 synthesis in neutrophils stimulated with other chemoattractants such as fmet-leu-phe and LTB4. We extended these studies to human monocytes and showed that addition of physiological concentrations of fibrinogen amplified IL8 production by monocytes as well as increased IL6 and TNF alpha production. In contrast, fibrinogen had no effect on monocyte chemoattractant protein1 (MCP1), inteferon beta, or interferon inducible protein10 (IP10). Treatment of monocytes with fibrinogen (less than 2 mg/ml) and complement 5 fragment, C5a, resulted in a 100% increase in both IL8 and IL6 production, compared to fibrinogen treatment alone. This was associated with a transient increase in monocyte IL8 mRNA and NF-&#954;B activity. Monocytes from patients with defective LPS and IL1 signaling through the Toll like receptor pathway (NEMO deficiency and IRAK4 deficiency) had 80% reduced IL8 response to fibrinogen compared with normal monocytes. Moreover, normal monocyte responses to fibrinogen were blocked by an antibody that blocks CD14, an important cofactor in TLR4-mediated LPS signal transduction. MY4 had no effect on cytokine production induced by PMA and ionomycin (Dough Kuhns).[unreadable] [unreadable] In 2008 we continued to study the molecular basis for priming and activation of the NADPH oxidase and superoxide production by endotoxin (LPS). The NADPH oxidase (NOX), an oligomeric enzyme, plays a key role in polymorphonuclear neutrophil (PMN)-mediated host defense by producing cytotoxic superoxide anion (O2.-). While in vitro and biochemical studies have examined the assembly and activation of this important host immune defense system, few studies have examined the function of NOX in human patients with primary immunodeficiencies other than chronic granulomatous disease. We studied the activation of NOX in PMN from patients with two distinct immunodeficiencies, interleukin-1 receptor associated kinase 4 (IRAK4) deficiency and nuclear factor kappa (NF-&#954;B) essential modulator (NEMO or IKK&#947;) deficiency. We observed impaired O2.- generation by LPS-treated and fMLP-activated IRAK4-deficient PMN that correlated with decreased phosphorylation of p47phox and subnormal translocation of p47phox, p67phox, Rac2, and gp91phox/Nox2 to the membranes indicating that TLR4 signaling to the NOX activation pathway requires IRAK4. NEMO-deficient PMN also generated less O2.- in response to LPS and fMLP and translocated less p47phox and p67phox to membranes than normal PMN but were more responsive than IRAK4-deficient cells. Decreased LPS and fMLP induced phosphorylation of p38 MAPK in both IRAK4- or NEMO-deficient PMN and of p21-activated kinases (PAK) in IRAK4-deficiency implicates additional signal transduction pathways in regulating PMN superoxide activation by LPS and fMLP (Anjali Singh, 85% effort; Kol Zarember 10% effort).